WO2025110671A1 - Novel strain having endurance-enhancing activity - Google Patents

Abstract

The present invention relates to a novel strain of Allobaculums (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) having anti-fatigue or endurance-enhancing activity, wherein the strain has the benefits of increasing endurance through an increase in exercise ability, increasing type 1 myofibers, biogenisis of mitochondria, and muscle development through biogenisis of muscle, and can be used for anti-fatigue, endurance enhancement, and exercise performance enhancement, and can also be used for preventing or treating muscle diseases.

Description

A new strain with endurance-enhancing activity

This application claims the benefit of Korean Patent Application No. 10-2023-0161520, filed on November 20, 2023, and Korean Patent Application No. 10-2024-0020587, filed on February 13, 2024, the entire contents of which are incorporated herein by reference.

The present invention relates to a new strain having endurance-enhancing activity, an anti-fatigue or endurance-enhancing composition comprising the same, and a composition for preventing or treating muscle diseases.

Muscles are largely divided into skeletal muscle, cardiac muscle, and visceral muscle. Among them, skeletal muscle accounts for 40% of the body weight, occupying a large portion of the human body, and together with bones, it maintains the shape of the human body and enables movement and movement. These bones and skeletal muscles are collectively called the musculoskeletal system. The basic cells of muscles are composed of myoblasts. When a muscle is injured or muscle tissue ruptures due to intense exercise, satellite cells, which are stem cells of muscle tissue, are activated and satellite cell division is promoted. Some of the increased satellite cells maintain the function of stem cells again, and some begin to differentiate into muscle cells. At this time, various transcription factors related to muscle cell differentiation are activated, and satellite cells differentiate into myoblasts, myocytes, and myotubes in that order, filling the injured area and damaged muscle tissue with new muscle fibers. Muscles are largely composed of two types of muscle fibers, of which type 1 muscle fibers contain many mitochondria and are essential for endurance exercise. However, in chronic metabolic diseases such as obesity and diabetes, which have increased due to Western-style eating habits and lack of exercise, type 1 muscle fibers decrease, causing endurance to decrease. Meanwhile, muscle growth can be largely divided into two types: hyperplasia, which increases the number of muscle cells and grows, and hypertrophy, which increases the size of cells and grows. Hyperplasia occurs mainly during the growth period, that is, from infancy to secondary sexual characteristics, and hypertrophy, which increases the size of muscles through exercise. In addition, if you continue to exercise appropriately, it stimulates muscle cells, and factors involved in muscle development are continuously expressed. The size and number of muscle fibers, muscle development, and muscle mass increase, as well as the biosynthesis and function of mitochondria, which synthesize ATP, which is important for muscle motility, are improved. Maintaining muscle homeostasis is essential for maintaining a smooth life, but in modern society, lack of exercise is occurring due to aging and changes in job distribution. As a result, muscle usage decreases, resulting in decreased muscle mass and muscle function, which can easily cause fatigue or lead to serious injuries from even minor collisions. In addition, behaviors harmful to the human body, such as irregular eating habits, stress, drinking, and smoking, also cause weakened muscle function. Muscle strength refers to the ability of muscles, and mainly refers to the maximum force that muscles can exert at one time. Exercise performance refers to the ability to perform exercise using muscle strength. Weakened muscle function and muscle weakness due to the above-mentioned problems lead to a decrease in muscle strength and exercise performance, which in turn causes decreased physical strength, increased adult diseases, and decreased quality of life.

Fatigue is defined as a state of reduced physical and mental activity ability, and physical fatigue (fatigue, establishment of a fatigue recovery-related functional evaluation system for health functional foods, 2004 Ministry of Food and Drug Safety research report), which refers to a state of reduced exercise performance ability, is caused by the accumulation of fatigue substances such as lactic acid in the muscles (Fitts et al., 1976; Savitski et al., 1979). Sufficient sleep and rest help recover from fatigue, but unrecoverable fatigue can cause various acute and chronic diseases. Fatigue can sometimes be defined as "a perception of reduced ability for physical and mental activity due to an imbalance in the availability, utilization, and recovery of resources necessary to perform an activity." Fatigue is mainly a state of reduced work ability due to physical fatigue, and stress is a state of mental fatigue that causes disruption of homeostasis. Fatigue is classified into central nervous system fatigue, fatigue of the neuromuscular junction, and peripheral fatigue of the limbs. Fatigue is a comprehensive physiological process including various physiological and biochemical factors, and is a mental physiological phenomenon that inevitably appears when the body's mental activity or physical activity reaches a certain level. It means that the body's original work ability is temporarily reduced, and it can be a symptom indicating that the body is developing into a damaged state. If fatigue occurs continuously and becomes chronic, it develops into a disease called Chronic Fatigue Syndrome (CFS). Currently, the main symptom of chronic fatigue syndrome is long-term fatigue, which is a syndrome that appears as a group of non-specific symptoms such as low-grade fever, headache, sore throat, muscle and joint pain, attention deficit disorder, memory loss, sleep disorder, and depression, and there are usually no significant abnormalities in physical examinations. There are various causes of fatigue, such as lack of sleep, lack of exercise, unbalanced diet, bad habits such as alcoholism, mental pressure, bad work environment, and workplace environment. There are various methods for relieving fatigue, including taking a rest, finding a satisfying job, regular exercise, changing to a better diet, and getting enough sleep. In addition, the market for functional products that focus on suppressing the accumulation of fatigue substances in the body to relieve physical fatigue and improving exercise performance is active, but products containing compounds such as steroids, caffeine, sodium bicarbonate, and sodium citrate, while able to significantly increase exercise performance when taken in doses exceeding a certain level, have the risk of causing fatal side effects and ultimately harming health. Recently, research is being actively conducted to develop products for improving fatigue and enhancing exercise performance using lactic acid bacteria with guaranteed safety.

The present invention has been devised to solve the above problems, and the inventors of the present invention have named a newly discovered new strain among the Allobaculum strains as Allobaculum lactocepinia , and have attempted to elucidate the characteristics of the Allobaculum strain and Bifidobacterium animalis .

The purpose of the present invention is to provide a strain of the genus Allobaculum having anti-fatigue or endurance-enhancing activity. (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) new strains are provided.

Another object of the present invention is to provide a strain of the genus Allobaculum (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the above strain, a lysate thereof and an extract thereof, the present invention relates to a pharmaceutical composition for preventing or treating muscle disease caused by muscle dysfunction, muscle wasting or muscle degeneration, the composition comprising at least one selected from the group consisting of a culture of the above strain, a lysate thereof and an extract thereof.

Another object of the present invention is to provide a strain of the genus Allobaculum The present invention relates to a method for preventing or treating muscle disease caused by decreased muscle function, muscle wasting or muscle degeneration, comprising a step of administering or ingesting to a subject a composition comprising at least one selected from the group consisting of Bifidobacterium animalis (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the above strain, a lysate thereof and an extract thereof.

Another object of the present invention is to provide a strain of the genus Allobaculum (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the above strain, a lysate thereof and an extract thereof, the composition comprising at least one selected from the group consisting of the above strain, a lysate thereof and an extract thereof, is used for the prevention or treatment of muscle disease caused by decreased muscle function, muscle wasting or muscle degeneration.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating at least one muscle disease selected from the group consisting of atony, muscular atrophy , muscular dystrophy, muscle degeneration, myasthenia , cachexia and sarcopenia, comprising at least one selected from the group consisting of Allobaculum genus strain (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof and an extract thereof.

Another object of the present invention is to provide a method for preventing or treating at least one muscle disease selected from the group consisting of atony , muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia and sarcopenia , comprising the step of administering or ingesting to a subject a composition containing at least one selected from the group consisting of a strain of the genus Allobaculum (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof and an extract thereof.

Another object of the present invention is to use a composition comprising at least one selected from the group consisting of a strain of the genus Allobaculum (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof, and an extract thereof, for the prevention or treatment of at least one muscle disease selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and sarcopenia.

One aspect of the present invention to achieve the above purpose is a strain of the genus Allobaculum having anti-fatigue or endurance-enhancing activity. (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) new strains.

In the present invention, the term Allobaculum genus strain lactocepinia (KCCM13405P) is a novel Allobaculum genus strain specifically isolated and identified by the inventors from the feces of mice treated with CBD, and is newly named Allobaculum lactocepinia in this patent, and can be used interchangeably with “genus strain” or “Allobaculum lactocepinia strain” .

In the present invention, the Allobaculum genus strain (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) has 16S rDNA consisting of the base sequences of sequence number 1 and sequence number 2. The strain is an Allobaculum genus strain. (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) and deposited at the Korean Culture Centre of Microorgamisms (KCCM) on October 17, 2023, and were assigned accession numbers KCCM13405P and KCCM13404P, respectively.

When the above strain was treated in mice, endurance significantly increased and the size of the gastrocnemius and soleus muscles increased. In the group treated with the above strain, the ratio of MyHC-Ⅱ, a specific gene related to slow muscle fibers, significantly increased. In the Bifidobacterium animalis administered group, the ratio of MyHC-Ⅱ related to fast muscle fibers significantly decreased. Groups A and B showed a significant increase in SDH-positive muscle fibers (Fig. 7). These results were also confirmed by mRNA expression, and were consistent with the changes in the expression of Type-Ⅱ, a marker protein of fast muscle fibers, and Type-Ⅱ, a marker protein of slow muscle fibers (Fig. 8). Through this, it was confirmed that the two strains improved endurance.

The lactate level of Bifidobacterium animalis significantly decreased, and the blood ketone bodies significantly increased in both strains. Since an increase in the blood ketone body concentration is an evaluation index of energy efficiency, and the lactate concentration is an index of an increase in muscle fatigue, it can be seen that the two strains contributed to the increase in energy efficiency, and in particular, Bifidobacterium animalis contributed to the decrease in muscle fatigue (Fig. 8).

In the present invention, the strain can increase the proportion of type 1 muscle fibers in muscle tissue.

In the present invention, the strain can increase the biogenesis of mitochondria in muscle tissue.

In the present invention, the strain can develop muscles through muscle neogenesis.

In the present invention, the strain can increase phosphorylation of AMPK or CREB and increase expression of PGC-1α.

Another aspect of the present invention is a strain of the genus Allobaculum The present invention relates to a pharmaceutical composition for preventing or treating muscle disease caused by decreased muscle function, muscle wasting or muscle degeneration, comprising any one selected from the group consisting of (KCCM13405P) and Bifidobacterium animalis (KCCM13404P) strains, cultures of the above strains, lysates thereof and extracts thereof.

In the present invention, the culture is a product obtained by culturing a microorganism in a medium, and the medium may be selected from known liquid media or solid media, and may be, for example, MRS liquid media, MRS agar media, or BL agar media.

The term "composition" used in the present invention means a material in which two or more components are uniformly mixed, and is a concept that includes not only a finished product but also an intermediate material for manufacturing a finished product.

The terms “pharmaceutically acceptable” and “foodstuff acceptable” as used in the present invention mean not significantly stimulating an organism and not inhibiting the biological activity and properties of the administered active substance.

The term “prevention” as used in the present invention means any act of suppressing symptoms or delaying progression of a specific disease by administering the composition of the present invention.

The term "treatment" as used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

The term "improvement" as used in the present invention means any action that at least reduces or alleviates a parameter related to the condition being treated, for example, the degree of a symptom.

The term "administration" used in the present invention means providing a given substance to a subject or patient by any appropriate method, and may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or locally as an injection formulation) or orally depending on the intended method, and the dosage may vary depending on the patient's weight, age, sex, health, diet, administration time, administration method, excretion rate, and disease severity. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and may include various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. The pharmaceutical composition of the present invention may be administered by any device capable of transporting an active substance to a target cell. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. The injection can be manufactured using aqueous solvents such as saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid ester (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and can include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

The term "subject" used in the present invention means all animals including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that have developed or may develop the muscle disease, and by administering the pharmaceutical composition of the present invention to the subject, the muscle disease can be effectively prevented or treated. The pharmaceutical composition of the present invention can be administered in parallel with existing therapeutic agents.

The term "pharmaceutically effective amount" in the present invention means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which may be determined based on factors including the type and severity of the disease of the subject, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field.

In another aspect of the present invention, a strain of the genus Allobaculum The present invention relates to a pharmaceutical composition for preventing or treating at least one muscle disease selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia and sarcopenia, comprising at least one selected from Bifidobacterium animalis (KCCM13405P) and Bifidobacterium animalis (KCCM13404P) strains, cultures of the above strains, lysates thereof and extracts thereof.

The above "atony" is a disease also called flaccidity or atonia, and refers to a state of decreased or lost muscle tone.

The above "muscular atrophy" refers to the loss of muscle tissue caused by not using the muscle, or disease of the muscle itself, or damage to the nerves that control the muscle, and includes both progressive muscle atrophy and muscle weakness. In general, there are cases where serious muscle strength loss occurs due to not using the muscle, and it gradually progresses to muscle atrophy, and in addition, in cases where people live in places without gravity or show symptoms of muscle strength decline due to a decrease in calcium and muscle strength.

In addition, it can include muscle atrophy due to disease of the muscle itself, such as myasthenia gravis, Duchenne, Becker, limb-girdle, and facioscapulohumeral types, and inflammation that occurs in the muscle itself, and it can also include spinal muscular amyotrophy (Berardnig-Hoffmann type, Kugelberg-Welander disease), amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), and spinobular muscular atrophy (Kennedy's disease) due to damage to the nerves that control the muscle.

The above "muscular dystrophy" is a type of degenerative muscle disease that causes necrosis of muscle fibers and causes muscle strength loss and atrophy through necrosis and degeneration of muscle fibers due to damage to the muscle cell membrane. These include Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and congenital muscular dystrophy, and can appear in various forms depending on the location.

In addition, the above muscle wasting or muscle degeneration may be caused by factors such as genetic factors, acquired factors, and aging, and may include all of the gradual loss of muscle mass, weakening and degeneration of muscles, especially skeletal muscles or voluntary muscles and cardiac muscles, and all of the resulting muscle weakness.

The above "muscle weakness" refers to a state in which the strength of one or more muscles is reduced. The muscle weakness may be limited to one muscle, one side of the body, the upper or lower limbs, or may occur throughout the body. In addition, subjective symptoms of muscle weakness, including muscle fatigue or muscle pain, can be objectively quantified through a medical examination. Causes of muscle weakness include, but are not limited to, muscle damage, muscle mass reduction due to decreased differentiation of muscle cells, and muscle aging.

The above "myasthenia" is a disease in which the muscles weaken due to a nerve disorder of the muscles. At first, mild symptoms such as drooping eyelids (ptosis) and poor eye movement appear. Also, when speaking, pronunciation is not accurate or food is not swallowed properly. In addition, facial muscles also become weak. When myasthenia becomes severe, the symptoms spread throughout the body, making it difficult to lift objects due to lack of strength in the arms and legs and easily falling. In addition, dangerous conditions such as shortness of breath and respiratory paralysis may appear.

The above "cachexia" refers to a state that brings about a marked general debility, and the causes include malignant tumor, Basedow's disease, hypopituitarism, malaria, hypoadrenocorticism, and thymic dysfunction. Clinically, the main symptoms are general debility, anemia, and edema. It is known that anemia of malignant tumor, malnutrition caused by local effects of the tumor itself, and systemically, cachexia occurs due to host metabolic disorders caused by metabolites of the tumor competing with the host's metabolism.

The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient or a combination of two or more thereof commonly used in biological preparations. The term "pharmaceutically acceptable" as used herein means that the composition exhibits a characteristic of not being toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, and for example, compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components may be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, a diluent, a dispersant, a surfactant, a binder and a lubricant may be additionally added to formulate the composition into an injection form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule or a tablet. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In the present invention, the pharmaceutical composition may be in one or more dosage forms selected from the group including oral dosage forms, topical preparations, suppositories, sterile injectable solutions, and sprays.

The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

Another aspect of the present invention relates to a composition for improving exercise performance, comprising any one selected from a culture of the strain, a lysate thereof, and an extract thereof.

In the present invention, the improvement in exercise performance ability may have at least one effect selected from the group consisting of increasing exercise endurance, strengthening muscle strength, improving balance sense, and improving exercise adaptation ability.

In the present invention, the “improvement of exercise performance ability” may mean preventing or treating one or more diseases selected from the group consisting of mitochondrial abnormalities, neurodegenerative diseases, decreased endurance, and decreased agility.

In the present invention, the “exercise performance ability” refers to the degree to which one can perform physical movements seen in daily life or sports, such as running, jumping, throwing, and swimming, quickly, strongly, accurately, for a long time, and skillfully. Exercise performance ability is defined by factors such as muscle strength, agility, and endurance, and “improvement in exercise performance ability” means improving or enhancing exercise performance ability.

In the present invention, the above "mitochondrial disease" is a disease caused by a dysfunction of mitochondria, and may include all diseases, such as a dysfunction caused by oxidative stress due to mitochondrial membrane potential abnormality, phosphate swelling, reactive oxygen species or free radicals, a dysfunction caused by genetic factors, such as mutations in mitochondrial DNA or nucleus genes related to mitochondrial function, and a disease caused by a defect in the oxidative phosphorylation function for energy generation of mitochondria.

The above mitochondria are essential cell organelles that produce ATP, which is cellular energy. If mitochondrial dysfunction occurs, the energy produced within the cell gradually decreases, and cell damage or even cell death follows. Accordingly, mitochondrial dysfunction impairs the functions of all cells that contain mitochondria, except for red blood cells that do not have mitochondria, and causes fatal damage in particular to organs with high energy demands, such as muscles and the brain. Mitochondrial dysfunction can affect virtually any tissue, and a wide variety of symptoms can exist depending on the degree of tissue involvement. Examples of specific mitochondrial dysfunction diseases include Friedreich's ataxia (FRDA), Leber's Hereditary Optic Neuropathy (LHON), and Dominant Optic atrophy (DOA); These include Mitochondrial Myopathy, Encephalopathy, Lactacidosis, Stroke (MELAS), Myoclonus Epilepsy Associated with Ragged-Red Fibers (MERRF) syndrome, Leigh syndrome, and oxidative phosphorylation disorders, and most mitochondrial diseases are known to cause neurodegenerative diseases, stroke, blindness, hearing impairment, diabetes, and heart failure.

Another aspect of the present invention relates to a food composition for preventing or improving muscle disease, comprising any one selected from a culture of the strain, a lysate thereof, and an extract thereof.

When the composition of the present invention is used as a food composition, the strain can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The composition can contain a food additive acceptable in terms of food science in addition to the effective ingredient, and the mixing amount of the effective ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment).

The term "food supplement additive" used in the present invention means a component that can be added to food as an auxiliary, and can be appropriately selected and used by those skilled in the art as added in the manufacture of health functional foods of each formulation. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like, but the types of food supplement additives of the present invention are not limited by the above examples.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients having useful functionality for the human body. Here, "functionality" means obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added to the food composition. In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time as it uses natural materials as raw materials, and is highly portable, so the health functional food of the present invention can be taken as a supplement to enhance the effect of a muscle disease treatment agent.

In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, the composition including the strain of the present invention can be manufactured by mixing other appropriate auxiliary ingredients and known additives that can be included in health functional foods according to the selection of a person skilled in the art. Examples of foods to which it can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and it can be manufactured by adding it to juice, tea, jelly, and juice manufactured using the extract according to the present invention as a main ingredient.

The Allobaculum genus strain ( KCCM13405P) and Bifidobacterium animalis (KCCM13404P) strain of the present invention increase endurance by increasing exercise capacity of mice, increase type 1 muscle fibers, enhance/improve mitochondrial neogenesis and function, and develop muscles through muscle neogenesis, and therefore can be used for anti-fatigue, endurance enhancement, and exercise performance enhancement purposes, and can also be used for the prevention or treatment of muscle diseases.

Figure 1 is a result showing increased exercise capacity in mice according to treatment with cannabidiol, (A) is a schematic diagram showing endurance exercise capacity measured on a treadmill by administering 30 mg/kg of cannabidiol for 4 weeks to 20-week-old C57/BL6 mice of the present invention, and (B-D) are the results of confirming endurance on a treadmill after exercise training on a treadmill for 4 weeks, in which (B) is the running time, (C) is the running distance, and (D) is the running time to fatigue.

Figure 2 shows the results measured using an indirect calorimeter according to treatment with cannabidiol or the strain of the present invention after changing the intestinal microbial community by administering cannabidiol or antibiotics.

Figure 3 shows the results of confirming the changes in the muscle fiber composition of the gastrocnemius muscle of mice according to cannabidiol administration. (A) is a representative result of skeletal muscle of a 20-week-old mouse according to treatment with a solvent or cannabidiol, (B) is the result of immunofluorescence staining with MyHC-I, MyHC-IIa, and MyHC-IIb targeting the gastrocnemius muscle, (C), (D), and (E) are the results of quantifying the composition and cross-sectional area of the muscle fibers using the images of the immunofluorescence staining, (F) is the result of confirming the changes in markers of slow and fast muscle fibers using q-PCR, (G) is the result of succinate dehydratase (SDH) staining of the gastrocnemius muscle, and (H) is the result of quantifying SDH-positive muscle fibers.

Figure 4 shows the results of confirming the change in mitochondrial biogenesis according to cannabidiol administration. (A) is the result of quantifying mitochondrial DNA (mtDNA) standardized to nuclear DNA (nDNA) by qPCR, (B) is the result of confirming genes related to mitochondrial biogenesis and its oxidative phosphorylation in the gastrocnemius muscle by qPCR, (C) and (D) are the results of representative Western blot analysis of the OxPhos complex and the results of quantifying the results, and (E) and (F) are the results of Western blot of genes related to mitochondrial biogenesis in the gastrocnemius muscle and the results of quantifying the results.

Figure 5 shows the results of confirming the change in the composition of intestinal microorganisms according to the administration of cannabidiol. (A) is the result of analyzing the effect of cannabidiol administration on the composition of intestinal microorganisms at the phylum level by synthetic sequence analysis, (B) is the result of analyzing the effect of cannabidiol administration on the composition of intestinal microorganisms at the family level by synthetic sequence analysis, (C) is the result of measuring beta diversity by analyzing the principal coordinate analysis (PCoA) plot using generalized weighted UniFrac as a distance measure, (D) is the result of measuring alpha diversity using Shannon and (E) is the result of measuring ACE index, and (F) is an outline of a study to confirm the effect of intestinal microorganism community on cannabidiol-mediated exercise performance enhancement. 20-week-old C57BL/6 mice were administered 40 mg/kg of doxycycline for 5 weeks after the treadmill adaptation period, and cannabidiol was administered for 4 weeks to measure endurance exercise performance on a treadmill. 30 mg/kg was administered, and (G-I) are the results of checking the endurance on the treadmill after 4 weeks of treadmill exercise training, where (G) is the running time, (H) is the running distance, and (I) is the running time to fatigue. (J) is the result of performing representative immunofluorescence staining for MyHC-I, MyHC-IIa, and MyHC-IIb/x of the gastrocnemius (GAS) muscle, (K) is the result of quantifying the muscle fiber composition of the mice in each group, (L) is a representative photograph of the representative succinate dehydrogenase (SDH) staining of the gastrocnemius muscle, and (M) is the result of quantifying this.

Figure 6 shows the results of confirming the change in mitochondrial biogenesis according to the administration of cannabidiol and antibiotics. (A) is the result of confirming the change in markers of slow and fast muscle fibers in the gastrocnemius muscle by q-PCR after treatment with solvent, cannabidiol, doxycycline, and co-administration of cannabidiol and doxycycline, respectively. (B) is the result of representative Western blot analysis of the OxPhos complex in each group. (C) is the result of Western blot of genes related to mitochondrial biogenesis in the gastrocnemius muscle. (D) is the result of quantifying the result. (E) and (F) are the results of confirming the genes related to mitochondrial biogenesis and its oxidative phosphorylation in the gastrocnemius muscle by qPCR. (G) is the result of analyzing the gut microbiota composition at the phylum level in each group by synthetic sequence analysis. (H) is the result of analyzing the gut microbiota composition at the family level by synthetic sequence analysis. (I) is the result of measuring alpha diversity using Shannon, (J) is the result of measuring beta diversity using ACE index, and (K) is the result of measuring beta diversity by analyzing principal coordinate analysis (PCoA) plot using generalized weighted UniFrac as a distance measure.

FIG. 7 shows the results of confirming the improvement in exercise capacity of mice after treatment with a solvent or cannabidiol or bacteria A, B, or F, wherein (A) is a schematic diagram of a study to confirm the improvement in exercise capacity according to the treatment of each strain, (B-D) are the results of confirming endurance on a treadmill after exercise training on a treadmill for 4 weeks, wherein (B) is the running time, (C) is the running distance, and (D) is the running time to fatigue, (E) is the result of representative skeletal muscles of mice (gastrocnemius and soleus muscles), (F) is the result of immunofluorescence staining with MyHC-I, MyHC-IIa, and MyHC-IIb targeting the gastrocnemius muscle, (G) is the result of quantifying the muscle fibers, composition, and cross-sectional area of the immunofluorescence staining image, (H) is the result of succinate dehydratase (SDH) staining of the gastrocnemius muscle, and (I) is the result of quantifying SDH-positive muscle fibers.

Figure 8 shows the results of measuring the changes in mice after treatment with solvent, cannabidiol, or bacteria A, B, or F. (A) shows the results of showing the change in body weight according to the treatment with each strain, (B) shows the results of analyzing the change in the concentration of blood short-chain fatty acids in mice according to the treatment with each strain, and (C) shows the results of confirming the change in markers of slow and fast muscle fibers in the gastrocnemius muscle by q-PCR.

Figure 9 shows the results of measuring changes in mice after treatment with solvent, cannabidiol, or bacteria A, B, or F, where (A) is the result of measuring changes in blood glucose levels, (B) is the result of measuring blood lactate concentration, (C) is the result of measuring blood ketone body concentration, (D) is the result of measuring alpha diversity using Shannon, (E) is the result of measuring ACE index, (F) is the result of analyzing gut microbiota composition at the phylum level by synthetic sequence analysis for each group, (G) is the result of analyzing gut microbiota composition at the family level by synthetic sequence analysis, (H) is the result of measuring beta diversity by analyzing principal coordinate analysis (PCoA) plot using generalized weighted UniFrac as a distance measure, and (I, J) are the results of showing the predicted chromosomes from whole genome analysis of bacteria A and B in different colors according to their composition and function, where (I) is bacteria B, named bacteria KBP-1, (J) is the result of strain A named KBP-1.

Figure 10 shows the results for identifying and analyzing bacteria A. (A) is the morphological result, (B) is a phylogenetic analysis graph performed based on the base sequence information of bacteria A, and (C) is the result of taxonomic comparison through whole genome analysis.

Figure 11 shows the results for identifying and analyzing bacteria B. (A) is the result of performing NCBI BLAST on the base sequence of bacteria B, and (B) is a phylogenetic analysis graph performed based on the base sequence information of bacteria A.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Confirmation of increased exercise capacity, composition of oxidative muscle fiber components, and mitochondrial biogenesis following cannabidiol treatment

Example 1.1 Confirmation of exercise ability according to treatment with cannabidiol

The present invention confirmed the exercise ability of mice by oral administration of cannabidiol to them through a single-lane treadmill. Mice were orally administered with a solvent (corn oil) or cannabidiol, and then involuntary exercise training was performed for 4 weeks using a single-lane treadmill (Fig. 1A). Specifically, cannabidiol dissolved in a solvent or corn oil at 30 mg/ml was orally administered to 20-week-old C57/BL6 mice 6 times a week. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 85-23, revised in 2011). The research protocol of the present invention was approved by the Institutional Animal Care and Use Committee of Chonbuk National University (Permit No.: JBNU 2022-048). Before evaluating exercise performance, mice were acclimated to a single-rail treadmill at a speed of 10 m/min for 30 minutes every day for 7 days. Mice were tested for exercise capacity on a single-rail treadmill at moderate intensity daily for 4 weeks. Moderate-intensity running was measured starting at a speed of 10 m/min for 10 min, which was increased by 2 m/min every 10 min, up to a maximum speed of 16 m/min until exhaustion. Exhaustion was defined as the inability to return to the single-rail treadmill despite a gentle stimulus with a wooden cane. The running time and distance for each mouse were recorded during the last 2 weeks of the running test period. As a result, it was confirmed that the endurance capacity represented by the average running time, running distance, and running time to exhaustion was significantly increased in mice treated with cannabidiol (Fig. 1B-Fig. 1D).

Metabolic capacity (energy intake and energy expenditure) was measured using an indirect caloric calculation system (Oxymax/CLAMS metabolic cage system). Before the experiment, mice were acclimated to the metabolic chamber for 2 days. Then, the oxygen consumption rate ( _VO2 ; mL/kg/h), carbon dioxide production rate ( _VCO2 ; mL/kg/h), and respiratory exchange ratio (RER; _VO2 / _VCO2 ) were measured. In mice treated with cannabidiol, RER ( _VO2 / _VCO2 ) was significantly reduced. This confirmed that the mitochondrial substrate preference shifted from glucose to fatty acids (Fig. 2).

1.2 Confirmation of changes in oxidative muscle fiber components according to treatment with cannabidiol

Enhanced endurance capacity is generally associated with increased oxidative muscle fibers. Therefore, to investigate the composition and cross-sectional area of muscle fiber types, several types of hindlimb muscles were analyzed in mice. Twenty-week-old C57/BL6 mice were orally administered cannabidiol dissolved in solvent (corn oil) or corn oil at 30 mg/mL six times a week, and then sacrificed after 4 weeks. Macroscopic observation after sacrifice revealed that the hindlimb muscles of cannabidiol-treated mice were red (Fig. 3A).

For staining of myosin heavy chain isoforms, serial muscle sections were preincubated in a blocking solution of stock goat serum. Primary MyHC antibodies (MyHC I (#BA-D5), MyHCIIa (#SC-71), and MyHCIIb (#BF-F3); DSHB, Iowa City, IA, USA) were incubated overnight at 4°C. After washing, secondary antibodies (Alexa Fluor 350-conjugated goat anti-mouse IgG2b (#A21140), Alexa Fluor 488-conjugated goat anti-mouse IgG1 (#A21121), Alexa Fluor 594-conjugated goat anti-mouse IgM (#A21044)) were used for 1 h at 37°C. For the specification of muscle fiber types, sections containing approximately 200 myofibers were selected. Images were acquired using a Leica DM750 microscope (Leica, Wetzlar, Germany). The muscle fibers were then manually classified as immunopositive or immunonegative. In addition, the cross-sectional area of the muscle fibers was calculated using iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany). Immunofluorescence staining for myosin heavy chain (MyHC) isoforms of the gastrocnemius (GAS) muscle revealed that cannabidiol administration increased the oxidative-myofiber density and decreased the size of the corresponding muscle fibers (Fig. 3B-Fig. 3E).

Total RNA was extracted from skeletal muscle tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was generated using primers provided in the cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA). Specific primers for each gene (Table 1) were designed using PrimerBank (https:/pga.mgh.harvard.edu/primerbank).

gene order MyHC-I(Myh7) Forward ACAAGCTGCAGCTGAAGGTG Reverse TCATTCAGGCCCTTGGCAC MyHC-IIa(Myh2) Forward CCAGCTGCACCTTCTCGTTTGCCAG Reverse CATGGGGAAGATCTGGTCTTCTT MyHC-IIb(Myh4) Forward CCTGGAACAGACAGAGAGGAGCAGGAGAG Reverse GTGAGTTCCTTCACTCTGCGCTCGTGC MyHC-IId(Myh1) Forward TGCAACAGTTCTTCAACCAC Reverse GCCAGGTCCATCCCAAAGT

[Table 1] qPCR reactions were performed in a final volume of 10 μL containing 10 ng of reverse-transcribed total RNA, 200 nM forward and reverse primers, and PCR master mix. qPCR was performed in 384-well plates using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). mRNA levels of each target gene were normalized to Gapdh (for nuclear-encoded genes) or 16S rRNA (for mtDNA-encoded genes). For analysis of mitochondrial DNA content, total DNA was extracted using a genomic DNA purification kit (Qiagen, Hiaden, Germany). Relative mtDNA was quantified by qPCR using primers for the mitochondrial-encoded gene cytochrome oxidase 2 (Cox2) normalized to the nuclear-encoded gene cyclophilin A (Ppia).

Comparing mRNA expression with previous results, MyHC-I (Myh7) and MyHC-IIa (Myh2), specific genes associated with slow muscle fibers in the gastrocnemius muscle, were significantly increased by cannabidiol treatment. At the same time, MyHC-IIb (Myh4), a specific gene associated with fast muscle fibers, was significantly decreased, while MyHC-IId (Myh1) showed no change (Fig. 3F).

After sacrifice, skeletal muscle tissues were placed in a 30% sucrose solution and embedded in liquid nitrogen-cooled isopentane. For succinate dehydrogenase (SDH) staining, cryosections (10 μm) of tissues were incubated in 0.2 M sodium phosphate buffer (pH 7.6) containing 0.6 mM nitro blue tetrazolium and 50 mM sodium succinate at 37°C for 30 min. Slides were washed with distilled water and mounted in aqueous mounting media. Immunostaining of muscle sections with succinate dehydrogenase (SDH), an indicator of mitochondrial activity, revealed that on average, more than 70% of muscle fibers were SDH-positive in cannabidiol-treated mice compared to an average of 60% in solvent-treated mice (Figs. 3G to 3H).

1.3 Confirmation of increased mitochondrial biogenesis following cannabidiol treatment

Since the increase in oxidative capacity of muscle fibers is related to mitochondrial biogenesis and its function, the mitochondrial content and the expression of genes related to it were confirmed in the gastrocnemius muscle of mice treated with cannabidiol. In addition, the expression of major transcription factors and regulatory factors involved in mitochondrial biogenesis was confirmed.

The increase in mitochondria in the gastrocnemius muscle was confirmed by quantifying the content of mitochondrial and nuclear DNA (Fig. 4A). The mitochondrial content was determined by the ratio of mitochondrial DNA to nuclear DNA (mtDNA/nDNA). To quantify the copy number of mitochondrial DNA (mtDNA), it was determined according to the method described in The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy. Autophagy. 2014; 10:785-802. Pyruvate kinase ( Pklr ) was used as a nDNA marker, and NADH dehydrogenase subunit 1 was used as a marker for mtDNA. Real-time PCR reactions were performed according to the manufacturer's instructions (QuantiFast SYBR Green PCR Master Mix; Qiagen, 204052), and temperature cycling was performed on an ABI Prism 7900HT Sequence Detection System. The mtDNA content was normalized to the nucleotide DNA content. Treatment with cannabidiol significantly increased the mitochondrial DNA (mtDNA) content (Fig. 4A). This was also confirmed by qPCR of related genes. Treatment with cannabidiol in the gastrocnemius muscle increased the expression of genes related to mitochondrial biogenesis and oxidative phosphorylation (Fig. 4B).

Western blot analysis was also performed to measure the changes in the expression of CⅠ~Ⅴ (ATP synthase) and related signaling proteins corresponding to various components of the electron transport chain complex in mitochondria. Cell or tissue homogenates (20 ug) were separated using 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were probed with primary antibodies against CREB (#9197), p-CREB (#9198) (Cell Signaling Technology, Beverly, MA, USA), T-OxPhos (ab110413) (Abcam, Cambridge, UK), HSP90 (#ADI-SPA-836-F, Enzo Life Sciences, Plymouth Meeting, PA, USA), and PGC-1α (#AB-3242, Millipore, Danvers, MA, USA). HSP90 was used as a loading control. After washing with PBS, the cells were incubated for 1 hour with horseradish peroxidase-conjugated IgG (Zymed, South San Francisco, CA, USA), and antibody signals were detected using a Las-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA). As a result, a significant increase was confirmed in response to cannabidiol administration (Fig. 4C to Fig. 4D).

Western blotting was performed for AMP-activated protein kinase (AMPK), Sirt1, and CREB for transcriptional activation of PGC-1α associated with the increase of mitochondria. As a result, cannabidiol administration increased the expression of Sirt1, p-AMPK, and p-CREB simultaneously with PKA activation, and significantly increased the expression of PGC-1α accordingly (Fig. 4E to Fig. 4F). In summary, it was confirmed that cannabidiol treatment improved muscle endurance by inducing conversion to slow muscle fiber type and mitochondrial biogenesis.

Example 2. Confirmation of changes in the composition of intestinal microorganisms following administration of cannabidiol

The composition of fecal microbiota of cannabidiol-treated mice was determined using 16S rRNA amplicon sequencing. Twenty-week-old C57/BL6 mice were orally administered cannabidiol dissolved in solvent (corn oil) or corn oil at 30 mg/mL six times a week, and feces were collected 4 weeks later. Genomic DNA (gDNA) samples were extracted from fecal samples using a commercial DNA isolation kit (QIAamp DNA StoolMini Kit). Amplification of gDNA was performed using a barcoded forward primer (515F: 5'-GTGCCAGCMGCCGCGGTAA-3') and a reverse primer (806R: 5'-GGACTACHVGGGTWTCTAAT-3') to target the V4 region of the bacterial 16S rDNA gene, and amplicon sequencing was performed on an Illumina iSeq 100 (San Diego, CA). In addition, a barcoded forward primer (341F: 5'-CCTACGGGGNGGCWGCAG-3') and a reverse primer (805R: 5'-GACTACHVGGGTATCTAATCC-3') to target the V3-V4 region of the bacterial 16S rDNA gene, and amplicon sequencing was performed. TRIMMOMATIC (ver. 0.39) was used to remove adapter sequences, and data quality control analysis was performed using Quantitative Insights Into Microbial Ecology (QIIME2, ver. 2022.02.). Chimeric sequences were removed by DADA2 via the q2-dada2 plugin, and diversity analysis was performed via the q2-diversity plugin. Taxonomies were identified for amplicon sequence variants (ASVs), which are feature classifiers of the RDP database. Microbial composition graphs, α-diversity plots, and principal coordinate analysis (PCoA) were visualized in R (ver. 4.2.2) and the ggplot2 package (ver. 3.4.2).

At the phylum level, cannabidiol treatment significantly increased the diversity of Bacillota and Actinomycetota (Fig. 5A). At the family level, cannabidiol treatment significantly increased the diversity of Erycipelotrichaceae and Bifidobacteriaceae , and decreased Oscillopiraceae, Bacteroidaceae , and Prevotellaceae (Fig. 5B). Shannon and Gini-Simpson indices representing α-diversity did not show significant changes, but principal coordinates analysis (PCoA) using a distance measure such as Uni-Frac weighting as a measure of β diversity confirmed that the two groups were significantly separated (Figs. 5C-5E). This confirmed that cannabidiol treatment affected the partitioning of microbial communities.

Example 3. Confirmation of a direct correlation between the exercise capacity-enhancing effect of cannabidiol and changes in the gut microbiota community

To investigate the direct correlation between the exercise capacity-enhancing effect of cannabidiol and the resulting changes in the gut microbiota, we conducted an intervention using antibiotic treatment. By comparing various types of antibiotics, we selected an antibiotic that caused minimal changes in the gut microbiota while eliminating Erycipelotrichaceae and Bifidobacteriaceae , which increase with cannabidiol administration. Doxycycline was selected to test how the gut microbiota changes when antibiotics were administered simultaneously before and after testing cannabidiol for exercise capacity (Fig. 5F). As a result, the average running time, running distance, and running time for fatigue in mice treated with cannabidiol significantly increased, whereas no changes were observed in the group treated with both antibiotics (doxycycline) and cannabidiol (Figs. 5G to 5I).

Indirect calorimetric analysis of energy metabolism also showed a significant decrease in RER (VCO ₂ /VO ₂ ) in mice administered cannabidiol, but there was no change in the group administered antibiotics and cannabidiol together. To further clarify the above results, immunofluorescence staining of MyHC isomers was performed in the gastrocnemius muscle. Unlike the effect of the cannabidiol alone treatment group, the combined treatment with antibiotics and cannabidiol did not show a decrease in oxidized muscle fiber density and corresponding fiber size (Figs. 5J to 5L). Comparing mRNA expression as in the previous results, cannabidiol treatment significantly increased Type-I, a specific gene associated with slow muscle fibers, in the gastrocnemius muscle, and significantly decreased Type-IIx, and antibiotics offset all of these effects (Fig. 5K). In immunohistochemical staining of muscle sections using SDH results, no increase in SDH-positive muscle fibers was observed in the group administered antibiotics and cannabidiol together (Figs. 5L to 5M).

Cannabidiol-induced oxidation (Fig. 6A) and mitochondrial CⅠ to CⅤ amounts (Fig. 6B) in the gastrocnemius muscle were offset. The increased expression of PGC-1α and its related Sirt1, AMPK, and p-CREB was also offset (Fig. 6C to 6D). Antibiotics offset the expression of genes related to mitochondrial oxidation induced by cannabidiol administration (Fig. 6E to 6F). Antibiotics controlled the proportion of the increased microbial community composition induced by cannabidiol administration and did not restore the gut microbial community pattern (Fig. 6G-K). In summary, we confirmed that the gut microbial changes induced by cannabidiol administration significantly contribute to the effect of improving exercise performance.

Example 4. Confirmation of the exercise capacity of bacteria A (KPB-2) and B (KPB-1) and confirmation of increase in oxidative muscle fibers

Based on the hypothesis that the exercise performance-enhancing effect of cannabidiol may be mediated through the direct effect of altered gut microbes, specific microorganisms that showed a significant increase after cannabidiol administration were selectively isolated. Allobaculum and Faecalibabulum Rodentium belonging to the Erysipelotrichaceae family and Bifidobacterium animalis belonging to the Bifidobacteriaceae family were included. Each microorganism was administered individually or as a consortium at the same ratio and compared with the exercise performance-enhancing effect of cannabidiol (Fig. 7A). The bacteria alone treatment groups were named after the initials of each strain. Group A was treated with Allobaculum strain (KBP-2), group B with Bifidobacterium animalis (KBP-1), and group F with Faecalibabulum Rodentium . And the mixed (MIX) group was treated with the three bacteria as a consortium at the same ratio.

As a result, endurance, represented by average running time, running distance, and running time to fatigue, significantly increased in groups A ( Allobaculum spp.; KBP-2 treatment group) and B ( Bifidobacterium animalis ; KBP-1 treatment group) but not in groups F (Faecalibabulum Rodentium treatment group) and MIX (Fig. 7B-Fig. 7D). When comparing the size and color of the gastrocnemius and soleus muscles upon visual observation after sacrifice, it was found that redness and size increased in the A ( Allobaculum spp.; KBP-2 treatment group), B ( Bifidobacterium animalis ; KBP-1 treatment group), and cannabidiol treatment groups (Fig. 7E).

The MyHC isomers of gastrocnemius muscle were compared through fluorescent staining of slow and fast muscle fibers. The MyHC-Ⅱ ratio associated with slow muscle fibers (type 1 muscle) significantly increased in the A, B, or cannabidiol treatment groups, and the MyHC-Ⅱ ratio associated with fast muscle fibers (type 2 muscle) significantly decreased in the B or cannabidiol treatment groups. The ratio of MyHC-Ⅰ increased only in the cannabidiol treatment group (Fig. 7F and Fig. 7G). SDH immunostaining showed a significant increase in SDH-positive muscle fibers in groups A and B, although not as much as in the cannabidiol treatment group (Fig. 7H and Fig. 7I). These results suggest that A ( Allobaculum spp.; KBP-2 treatment group) and B ( Bifidobacterium animalis ; KBP-1 treatment group) promoted the development of type 1 muscle (slow muscle fibers; oxidative muscle fibers), thereby enhancing endurance exercise performance such as running.

These results were also confirmed by mRNA expression, and were consistent with the changes in the expression of Type-II, a marker protein of fast muscle fibers, and Type-II, a marker protein of slow muscle fibers. Among other slow fiber marker proteins, Tnni1 and Tnnt1 significantly increased only in the cannabidiol treatment group, and among the fast fiber marker proteins, Type-ⅠⅠTnni2, Tnnc2, and Tnnt3 decreased particularly only in the cannabidiol treatment group (Fig. 8C).

Example 5. Confirmation of changes in energy metabolism and intestinal microbiota communities of bacteria A and B

There was no significant difference in blood sugar levels, but lactate levels significantly decreased in group B and the cannabidiol-administered group, and blood ketone bodies significantly increased in groups A, B, and cannabidiol-administered (Fig. 9A-Fig. 9C). When the increase in blood ketone body concentration is evaluated as an increase in energy efficiency, and the lactate concentration is evaluated as an increase in muscle fatigue, it can be seen that bacteria B and cannabidiol contributed to the increase in energy efficiency and the decrease in muscle fatigue.

Analysis of changes in the composition of the gut microbiota showed that there was no effect on alpha diversity (Figs. 9D and 9E), but there were differences in composition (Figs. 9F and 9G), and PCoA using weighted UniFrac showed a significant distance between the groups (Fig. 9H). At the family level, Erysipelotrichaceae increased in treatment group A, Bifidobacteriaceae increased in treatment group B, and both increased in the cannabidiol treatment group (Figs. 9F and 9G). Bacteria A and B that showed a significant effect of improving muscle endurance were subjected to whole-genome analysis (Figs. 9I and 9J). It was confirmed that gut microbiota A and B species increased by cannabidiol administration affected exercise performance and energy metabolism improvement through changes in the microbial community and microbial effects even without the effect of absorbed cannabidiol.

Example 6. Identification through base sequence analysis of bacteria A and B

In Examples 4 and 5 above, Allobaculum species belonging to the Erysipelotrichaceae family and Bifidobacterium animalis strains belonging to the Bifidobacteriaceae family were analyzed and identified as being effective in anti-fatigue and enhancing endurance.

6.1 Belonging to the Erysipelotrichaceae family Allobaculum Analysis and identification of the inside

To isolate Allobaculum genus in the family Erysipelotrichaceae, CBD-treated mouse feces (0.1 g) were immediately collected, suspended and diluted in 0.9 mL of general anaerobic medium, and streaked onto glucose blood liver agar plates containing 5% sheep blood. After streaking, the plates were incubated under anaerobic conditions at 37°C for 2 days in an anaerobic chamber with a GasPak EZ anaerobic pouch. Surface colonies were convex, translucent, and smooth. Bacteria cultured in GAM liquid medium at 37°C under anaerobic conditions for 2 days were subjected to Gram staining to observe their morphology as previously reported. Morphological observation was performed using an optical microscope, and they exhibited characteristics of Gram-positive, rod-shaped bacteria (Fig. 10A). The width was estimated to be about 1 μm and the length was 1–2 μm.

Phylogenetic analysis of the above strains was performed based on 16S rRNA sequences. The 16S rRNA sequence-based phylogenetic tree was performed based on the description of Paster et al. The Neighbor Join method, also known as the Agglomerative Clustering method, was used in MEGA 11.0.8 software, and the bootstrap test was performed based on 1,000 resamplings. The 16S rDNA sequences were obtained from the whole genome sequencing results in the fasta file, and the list of comparable strains was obtained from the GeneBank database using the nucleotide BLAST program of the National Center for Biotechnology Information (Fig. 10B). The 16S rRNA sequence is deposited in GenBank with the National Center for Biotechnology Information (NCBI) accession number PP082459.

According to the manufacturer's instructions, genomic DNA (gDNA) of the strains was extracted with the MagAttract HMW DNA Kit. The size, purity, and quality-controlled gDNA was purified with AMpureXP beads and constructed into the SMRTbell library using the SMRTbell Express Template Prep Kit PacBio, catalog number 100-93-8-9-00. After AMPure purification, the sequence information was deposited on the SMRT Cell 1M v2 sequel (Pacific Biosciences) platform using the Sequel Sequencing Kit v3.0. The nucleotide sequence of the genus Allobaculum in the family Erysipelotrichaceae was performed by CJ Bioscience, Inc. using PacBio sequencing data, and the data were assembled with SMRT Link using the protocol of Microbial Assembly (Pacific Biosciences, USA). Protein coding sequence (CDS) analysis, gene prediction and annotation such as coding or noncoding RNA, and heterologous group classification were performed using the UBLAST program with Prodigal 2.6.2, tRNAscan-SE 1.3.1, Rfam 12.0, EggNOG 4.5, Swissprot, KEGG, and SEED databases. Whole genome sequencing was analyzed to understand the genetic characteristics of the genus Allobaculum in the family Erysipelotrichaceae. To analyze the similarity with other strains at the whole genome level, the EzBioCloud Whole Genome public database and the Orthologous Average Nucleotide Identity (OrthoANI) analysis algorithm were used to select five strains putatively similar to Allobaculum in the family Erysipelotrichaceae. The phylogenetic results through OrthoANI analysis and the complete calculation of pairwise orthogonality detection of the selected strains were used to color-code the similarity between the matched sequences in the pairwise orthogonality matrix (POM) table (Fig. 10C).

The whole genome of the genus Allobaculum in the family Erysipelotrichaceae was analyzed to search for homology. It showed the highest homology to strains of the genus Allobaculum [phylum Bacillota; class Erysipelotrichia; order Erysipelotrichales; family Erysipelotrichidae] and appeared to form a single lineage with them. However, since the similarity with the 16S rRNA sequence of previously known strains was less than 95%, it was determined to be a new strain and was newly named (Fig. 10B). Based on these results, it was confirmed to be a new strain belonging to the genus Allobaculum and was named Allobaculum lactocepinia .

6.2 Bifidobacterium animalis Analysis and identification of

To isolate Bifidobacterium animalis , the feces (0.1 g) of CBD-treated mice were immediately collected, suspended and diluted in 0.9 mL of general anaerobic medium, and streaked onto glucose blood liver agar plates containing 5% sheep blood. After streaking, the plates were incubated under anaerobic conditions at 37°C for 2 days in an anaerobic chamber with a GasPak EZ anaerobic pouch. Surface colonies were convex, translucent, and smooth. The bacteria cultured in GAM liquid medium at 37°C under anaerobic conditions for 2 days were subjected to Gram staining to observe their morphology, as previously reported. Morphological observation was performed using an optical microscope, and it exhibited characteristics of Gram-positive, rod-shaped bacteria. The width was estimated to be approximately 1 μm and the length was 1–2 μm.

Phylogenetic analysis based on 16S rRNA sequences of the above strains was performed. The phylogenetic tree based on 16S rRNA sequences was performed based on the description of Paster et al. The Neighbor Join method, also known as the Agglomerative Clustering method, was used in MEGA 11.0.8 software, and the bootstrap test was performed based on 1,000 resamplings. The 16S rDNA sequences were obtained from the whole genome sequencing results of the fasta file, and the list of comparative strains was obtained from the GeneBank database using the nucleotide BLAST program of the National Center for Biotechnology Information (Fig. 11). As a result, it was confirmed that bacteria B was Bifidobacterium animalis .

While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

[Acceptance number]

Name of depositor: Korea Center for Microbiological Conservation (KCCM)

Accession number: KCCM13405P

Date of Consignment: 20231017

Name of depositor: Korea Center for Microbiological Conservation (KCCM)

Accession number: KCCM13404P

Date of Consignment: 20231017

Claims (8)

A new strain of Allobaculums genus (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) having anti-fatigue or endurance-enhancing activity. In claim 1, the strain is a novel strain that increases mitochondrial biogenesis. In the first paragraph, the strain is a novel strain that increases the proportion of type 1 muscle fibers in muscle tissue. A pharmaceutical composition for preventing or treating muscle disease caused by muscle dysfunction, muscle wasting or muscle degeneration, comprising at least one selected from the group consisting of the strain of claim 1, a culture of the strain, a lysate thereof and an extract thereof. A pharmaceutical composition for preventing or treating at least one muscle disease selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia and sarcopenia, comprising at least one selected from the group consisting of the strain of claim 1, a culture of the strain, a lysate thereof and an extract thereof. A pharmaceutical composition for preventing or treating muscle disease, which develops muscles through muscle regeneration, in claim 4 or 5. A composition for improving exercise performance, comprising any one selected from the strain of claim 1, a culture of the strain, a lysate thereof, and an extract thereof. A composition for improving exercise performance in claim 7, wherein the improvement in exercise performance has at least one effect selected from the group consisting of increasing exercise endurance, strengthening muscle strength, improving balance, and improving exercise adaptation.

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